Tissues of the clawed frog Xenopus laevis contain two closely related forms of UDP-GlcNAc:alpha3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I.

UDP-GlcNAc:alpha3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GnTI; EC 2.4.1.101) is a medial-Golgi enzyme that is essential for the processing of oligomannose to hybrid and complex N-glycans. On the basis of highly conserved sequences obtained from previously cloned mammalian GnTI genes, cDNAs for two closely related GnTI isoenzymes were isolated from a Xenopus laevis ovary cDNA library. As typical for glycosyltransferases, both proteins exhibit a type II transmembrane protein topology with a short N-terminal cytoplasmic tail (4 amino acids); a transmembrane domain of 22 residues; a stem region with a length of 81 (isoenzyme A) and 77 (isoenzyme B) amino acids, respectively; and a catalytic domain consisting of 341 residues. The two proteins differ not only in length but also at 13 (stem) and 18 (catalytic domain) positions, respectively. The overall identity of the catalytic domains of the X. laevis GnTI isoenzymes with their mammalian and plant orthologues ranges from 30% (Nicotiana tabacum) to 67% (humans). Isoenzymes A and B are encoded by two separate genes that were both found to be expressed in all tissues examined, albeit in varying amounts and ratios. On expression of the cDNAs in the baculovirus/insect cell system, both isoenzymes were found to exhibit enzymatic activity. Isoenzyme B is less efficiently folded in vivo and thus appears of lower physiological relevance than isoenzyme A. However, substitution of threonine at position 223 with alanine was sufficient to confer isoenzyme B with properties similar to those observed for isoenzyme A.

Modulation of invasive properties of murine squamous carcinoma cells by heterologous expression of cathepsin B and cystatin C.

Murine SCC-VII squamous carcinoma cells have the capacity to penetrate reconstituted basement membranes (Matrigel) in vitro. The invasion of Matrigel layers by SCC-VII cells was significantly reduced by E-64, a specific inhibitor of lysosomal cysteine proteinases. The cathepsin-B-selective E-64 derivative, CA-074, inhibited penetration of Matrigel by SCC-VII cells to the same extent, indicating a major role for this particular lysosomal enzyme in extracellular-matrix degradation during squamous-carcinoma-cell invasion. SCC-VII cells were stably transfected with a cDNA encoding human procathepsin B, in an attempt to modulate the invasive properties of the cell line. The transfected cells expressed the heterologous gene, secreted increased amounts of procathepsin B and displayed enhanced invasive potential. In vivo, the activity of cathepsin B is strictly regulated by endogenous inhibitors. SCC-VII cells were therefore also stably transfected with a cDNA encoding human cystatin C, the most potent cysteine-proteinase inhibitor in mammalian tissues. The expression of this transgene resulted in the production of active recombinant cystatin C and a pronounced reduction in Matrigel invasion. These studies demonstrate that the invasive properties of squamous-cell carcinomas can be changed by modulation of the balance between cathepsin B and its endogenous inhibitors, and provide further evidence for the involvement of this lysosomal cysteine proteinase in tumour invasion and metastasis.

Molecular cloning and characterization of cDNA coding for beta1, 2N-acetylglucosaminyltransferase I (GlcNAc-TI) from Nicotiana tabacum.

In plants as well as in animals beta1, 2N-acetylglucosaminyltransferase I (GlcNAc-TI) is a Golgi resident enzyme that catalyzes an essential step in the biosynthetic pathway leading from oligomannosidic N-glycans to complex or hybrid type N-linked oligosaccharides. Employing degenerated primers deduced from known GlcNAc-TI genes from animals, we were able to identify the cDNA coding for GlcNAc-TI from a Nicotiana tabacum cDNA library. The complete nucleotide sequence revealed a 1338 base pair open reading frame that codes for a polypeptide of 446 amino acids. Comparison of the deduced amino acid sequence with that of already known GlcNAc-TI polypeptides revealed no similarity of the tobacco clone within the putative cytoplasmatic, transmembrane, and stem regions. However, 40% sequence similarity was found within the putative C-terminal catalytic domain containing conserved single amino acids and peptide motifs. The predicted domain structure of the tobacco polypeptide is typical for type II transmembrane proteins and comparable to known GlcNAc-TI from animal species. In order to confirm enzyme activity a truncated form of the protein containing the putative catalytic domain was expressed using a baculovirus/insect cell system. Using pyridylaminated Man(5)- or Man(3)GlcNAc(2)as acceptor substrates and HPLC analysis of the products GlcNAc-TI activity was shown. This demonstrates that the C-terminal region of the protein comprises the catalytic domain. Expression of GlcNAc-TI mRNA in tobacco leaves was detected using RT-PCR. Southern blot analysis gave two hybridization signals of the gene in the amphidiploid genomes of the two investigated species N. tabacum and N.benthamiana.

Recombinant soluble low density lipoprotein receptor fragment inhibits minor group rhinovirus infection in vitro.

A fragment of the low density lipoprotein receptor encompassing the seven ligand binding repeats was expressed in Sf9 insect cells as a fusion protein with a carboxyl-terminally linked hexa-his tag by using a baculovirus vector. Up to 10 mg/l of the fusion protein was secreted into the medium. The material was soluble in the absence of detergent and active in binding beta very low density lipoprotein and a member of the minor group of human rhinoviruses (HRV2) in ligand blots from sodium dodecyl sulfate-polyacrylamide gels run under nonreducing conditions. The receptor fragment specifically inhibits viral infection of HeLa cells by minor group HRVs in a concentration-dependent manner. Viral infectivity is neutralized by aggregation.

Long-lived Th2 clones specific for seasonal and perennial allergens can be detected in blood and skin by their TCR-hypervariable regions.

We investigated the longevity of allergen-specific Th cells derived from patients suffering from either allergic rhinitis or atopic dermatitis. T cell clones (TCC) specific for seasonal and perennial allergens were raised. To determine whether these TCC were long-lived in vivo, PBMC and allergen-specific polyclonal T cell lines, collected and established inside a period of up to 4 years, were screened for the TCC of interest. For this purpose, a T cell tracing protocol was established in which oligonucleotides specific for the TCR beta-chain hypervariable junctional region were used as tools to identify each particular TCC. Seven pollen-specific TCC and two house dust mite-specific TCC, with a Th2-like cytokine production pattern in vitro, were demonstrated to be long-lived memory T cells in vivo. Specificity of the tracing protocol was ascertained by TCR sequence analysis. We conclude that allergen-specific TCC can persist for years, evidence for which can be monitored in blood, but also in the target organ of the allergic disorder. The data indicate that in vitro-characterized, allergen-specific, long-lived TCC may well reflect a repertoire of T lymphocytes of pathogenetic importance in vivo.

Recombinant soluble low-density lipoprotein receptor fragment inhibits common cold infection.

A fragment of the human low-density lipoprotein receptor encompassing the seven ligand-binding repeats fused to a C-terminal oligo-His tag was expressed in Sf9 insect cells. The melittin signal sequence encoded in the baculovirus vector led to secretion of the protein into the cell supernatant in a soluble form. The receptor fragment bound its natural ligand beta-migrating very-low-density lipoprotein and human rhinovirus serotype 2 in non-reducing ligand blots. Infection of all minor group human rhinovirus serotypes investigated was inhibited by the presence of the receptor fragment during viral challenge of HeLa cells. Infection is inhibited by aggregation of the virions.

Insect cells contain an unusual, membrane-bound beta-N-acetylglucosaminidase probably involved in the processing of protein N-glycans.

The beta-N-acetylglucosaminidase activity in the lepidopteran insect cell line Sf21 has been studied using pyridylaminated oligosaccharides and chromogenic synthetic glycosides as substrates. Ultracentrifugation experiments indicated that the insect cell beta-N-acetylglucosminidase exists in a soluble and a membrane-bound form. This latter form accounted for two-thirds of the total activity and was associated with vesicles of the same density as those containing GlcNAc-transferase I. Partial membrane association of the enzyme was observed with all substrates tested, i.e. 4-nitrophenyl beta-N-acetylglucosaminide, tri-N-acetylchitotriose, and an N-linked biantennary agalactooligosaccharide. Inhibition studies indicted a single enzyme to be responsible for the hydrolysis of all these substrates. With the biantennary substrate, the beta-N-acetylglucosaminidase exclusively removed beta-N-acetylglucosamine from the alpha 1,3-antenna. GlcNAcMan5GlcNAc2, the primary product of GlcNAc-transferase I, was not perceptibly hydrolyzed. beta-N-Acetylglucosaminidases with the same branch specificity were also found in the lepidopteran cell lines Bm-N and Mb-0503. In contrast, beta-N-acetylglucosaminidase activities from rat or frog (Xenopus laevis) liver and from mung bean seedlings were not membrane-bound, and they did not exhibit a strict branch specificity. An involvement of this unusual beta-N-acetylglucosaminidase in the processing of asparagine-linked oligosaccharides in insects is suggested.

Activation of procathepsin B in human hepatoma cells: the conversion into the mature enzyme relies on the action of cathepsin B itself.

In order to elucidate the processing mechanism of the lysosomal cysteine proteinase, cathepsin B, in mammalian cells, recombinant rat and human cathepsin B precursors were expressed in Saccharomyces cerevisiae. The active-site cysteine residue was changed to serine to prevent autoprocessing. When the purified proenzymes were incubated with the soluble fraction of postnuclear organelles obtained from human hepatoma HepG2 cells, processing to a 33 kDa form corresponding to the mature endogenous single-chain enzyme was observed. Inhibitors of metallo-, serine and aspartic proteinases exerted no significant effect on procathepsin B processing in vitro. However, the processing activity was effectively blocked by cysteine proteinase inhibitors, in particular E-64 and its cathepsin-B-selective derivative CA-074. Processing positions were identified by using anti-peptide antibodies specific for epitopes in the N- and C-terminal cleavage regions. The single-chain form produced in vitro was thus shown to contain an N-terminal extension of at least four residues relative to the mature lysosomal enzyme, as well as a C-terminal extension present in the proenzyme but usually absent in fully processed cathepsin B. On expression of the wild-type proenzyme in yeast, procathepsin B undergoes autoprocessing, yielding a single-chain form of the active enzyme, which contains similar N- and C-terminal extensions. These results indicate that maturation of procathepsin B in vivo in mammalian tissues relies on the proteolytic activity of cathepsin B itself.